1. Field of the Invention
The present invention relates generally to protein and genetic engineering, and more specifically to a method for in situ construction of mutagenesis libraries and kits used therefor.
2. Description of the Related Art
Directed evolution methods are increasingly used on industrial enzymes to improve the enzymes' substrate specificity, activity, thermostability, and high-temperature activity etc. (1-4). The success of directed protein evolution experiments hinges on the efficiency of methods used to create random mutagenesis libraries and screen libraries for mutants with properties of interest (5). Because library diversity represents a crucial parameter in directed evolution of a target gene for improved functionality, various protocols have been established for creating random mutagenesis libraries (6). Random mutagenesis, along with genetic selection or high-throughput screening (5), constitutes an important approach to identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties.
Many methods for directed evolution of enzymes were reviewed by Lutz and Patrick (7); and some of the more recent in vitro DNA mutagenesis approaches were described in both polymerase chain reaction (PCR) and non-PCR categories (8). One of the most commonly used random mutagenesis methods is error-prone PCR (9), which introduces random mutations during PCR by reducing the fidelity of the DNA polymerase. The natural error rate of the polymerase can be altered and enhanced by modifying the standard PCR methods (10). This technique has the advantage of developing new enzymatic properties without a structural understanding of the targeted enzyme, and often yields unique mutations that could not be predicted (10). Early techniques of error-prone PCR generally involves the following steps: 1) amplifying the target gene as the PCR template under error-prone conditions, to generate amplified target sequence that contain random mutations; 2) treating the terminal of the amplified target sequences using restriction endonucleases; 3) ligating the treated target sequence into a suitable expression vector using a DNA ligase; and 4) transforming the expression vector containing the target sequences into a suitable host cell, to obtain a population of cells, or mutagenesis library, which contain the various target sequences. This process is similar to a cloning or subcloning process of the target sequence, except that in conventional cloning or subcloning, only a small number of transformants need to be obtained, while in the construction of a mutagenesis library, generally tens of thousands of transformants are needed in order to realistically be able to obtain a suitable target mutant. This is a tedious and inefficient process.
In order to improve efficiency, a method of PCR amplification of whole plasmid using mega-primer, referred to as “MEGAWHOP” was devised for generating random mutagenesis libraries (11). This process effectively modifies steps 2 and 3 of the above process, and uses randomly mutagenesized target sequence as PCR primers, expression vector as the template, and high-fidelity DNA polymerase. Amplification products are treated with Dpn I, which degrades the template expression vector, but leaves intact the amplified product. The process yields double-stranded full-strength plasmid, avoiding the steps of restriction enzyme treatment and ligation. This method yields libraries that are virtually free of plasmid containing no or multiple inserts. The mega-primer-based PCR method has been improved greatly since it was originally developed (12-15).
Nevertheless, the mega-primer-based PCR method has its own drawbacks. For example, the mutated target sequences have to be specifically synthesized for each target sequence. Further, for the process to be somehow satisfactory, the size of the megaprimers should be in the range of between 500 to 1000 bases, thereby limiting the size of the target sequence. The amplification products using the mega primers are linear sequences, which is not amenable to forming a circular plasmid having two nicks, resulting in relatively low transformation efficiency. Furthermore, the method of mutagenizing the megaprimers also involves a restriction enzyme digestion of the PCR product and of the expression vector, making the process complicated and low in efficiency.
Therefore, there is a need for a faster, simpler and more universally applicable method for generating random mutagenesis libraries.